This is the second Project in a four Project group which is examining multiple aspects of mitochondrial functioning and mitochondrial defects associated with Parkinson's Disease (PD), a major neurodegenerative disease afflicting at least 1 million Americans. Project 2 is focused upon the pathogenetic mechanisms leading to the progressive neuronal cell death of the neurons affected in PD. The study will be conducted using cybrid model neurons and primary neurons in culture. Cybrid model neurons are constructed from a clonal human neural cell line lacking mitochondrial DNA (mtDNA) and patient-derived mitochondria. The project will examine a single central hypotheses: Mitochondrial involvement in oxidative stress, Ca2+ homeostasis and regulatory signaling is linked to increased vulnerability in Parkinson's disease- derived cybrids (PD cybrids). Mitochondria play an important role in the maintenance play an important role in the maintenance and efficacy of the calcium signaling network, especially in neurons. This is reflected in specific morphological and functional relationships between mitochondria and other cellular elements in the network, including the endoplasmic reticulum. The calcium signaling network plays a critical role coordinating exocytotic secretion, cellular metabolism, gene expression and inter-organelle communication. Mitochondria also function as a defense against cytotoxic of excitotoxic events and are implicated as a critical source of apoptotic signals including release of cytochrome c. Experiments will investigate the relationship between altered vulnerability to cell death in the cybrids derived from patients with Parkinson's disease and altered mitochondrial functioning in the calcium signaling network by addressing several aims: 1) What quantitative relationship exists between defects in ETC complex I activity, Ca2+ homeostasis and vulnerability to cell death in cybrids? Does neuroprotection involve restoration in normal Ca2+ homeostasis? 2) Does the impact of long-term inhibition of ETC complex I activity differ from that of acute inhibition? With respect to Ca2+ homeostasis? Cell death? Generation of oxyradicals? Mitochondrial morphology and inter- organelle interactions? 3) How are morphological and functional relationships of mitochondrial of mitochondria to other organelles affected in the PD cybrids? 4) How is mitochondrial movement altered in PD cybrids and in primary neurons after chronic rotenone treatment? 5) How do the mitochondrial defects in PD cybrids affect lysosomal activity, protein ubiquitination, degradation and the formation of inclusions that resemble Lewy bodies? The results gained will reveal novel aspects of the probable pathogenesis of this important disease in a carefully controlled model systems and may reveal therapeutic targets.